The focus of this proposal is the characterization of the transcription factor PU. 1. This factor belongs to a growing family of transcription factors that are related based on a high degree of sequence identity within an 85 amino acid region. This region serves as the DNA binding domain for these proteins and has been named the ETS domain. The original member of this family was v-ets, which was part of a 135 kD fusion protein expressed from the avian leukemia virus E26. This virus has been linked to the development of both erythroid and myeloid leukemias in chickens. Other members of this family have also been linked to the development of leukemias, including PU.1 and Fli-1. These results strongly suggest that many of the ETS domain proteins are involved in the control of cell proliferation. The PU.1 protein is expressed primarily in macrophages and B cells, and binds to a purine rich sequence having a core sequence of 5' - GGAA-3'. PU.1 has been shown to be a transcriptional activator and may be involved in the expression of the immunoglobulin light and heavy chain genes as well as the integrin CD11b. The overall design of this application is to define the role of PU. 1 in the regulation of transcription and identify genes that are regulated by PU. 1. The first specific aim is to characterize in detail the activation, protein-protein interaction and the DNA binding domains. Mutation of specific amino acids will be used to define the domains more clearly. The second specific aim is to disrupt the PU.1 gene in ES cells and in mice by homologous recombination. Results from these experiments will help define the role of PU.1 in the differentiation and growth of macrophages and B cells. We will also look for genes that may be regulated by PU.1. The third specific aim is to characterize the promoter and possible enhancer of PU.1. We propose to identify the cis acting elements within the promoter and characterize the proteins that bind to these elements. We are particularly interested in whether PU. 1 is involved in the regulation of itself and which of the DNA binding proteins involved in PU.1 expression are macrophage or B cell specific. The long range goals of the project are to gain a better understanding of the PU.1 transcription factor as well as the family of ETS domain transcription factors. It is likely that these factors play an important role in the development of cell lineages within the hematopoietic system.